Hepatoprotective activity of Mammea africana ethanol stem bark extract

Objective: The stem bark of Mammea africana Sabine (Guttiferae), (M. africana) a common plant that has been traditionally used to treat various diseases and ailments was evaluated for hepatoprotective potentials against paracetamol-induced liver injury in rats. Materials and Methods: The hepatoprotective effect of the stem bark extract (30-90 mg/kg) was evaluated by the assay of liver function parameters, namely total and direct bilirubin, serum protein and albumin, total cholesterol, alanine aminotransaminase (ALT), aspartate aminotransaminase (AST), and alkaline phosphatase activities (ALP), antioxidant enzymes: superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), reduced glutathione (GSH) and histopathological study of the liver. Results: Administration of the stem bark extract caused a significant (p<0.05 – 0.001) dose-dependent reduction of high levels of liver enzymes (ALT, AST and ALP), total cholesterol, direct and total bilirubin as well as elevation of serum levels of total protein, albumin and antioxidant enzymes (SOD, CAT, GPx and GSH). Histology of the liver sections of extract and silymarin-treated animals showed reductions in the pathological features compared to the paracetamol-treated animals. The chemical pathological changes were consistent with histopathological observations suggesting marked hepatoprotective effect of the stem bark extract of M. africana. Conclusion: The results show that the stem bark extract of M. africana has hepatoprotective potential which may be due to its antioxidant activity.


Plant collection
The plant material M. africana (stembark) was collected in April 2013 from a forest in Uruan area, Akwa Ibom State, Nigeria. The plant was identified and authenticated by Dr. Magaret Bassey, Department of Botany and Ecological Studies, University of Uyo, Uyo, Nigeria. Herbarium specimen was deposited at the hebarium of Faculty of Pharmacy with voucher no. FPHUU 381.

Extraction
The pieces of the stembark were washed and shade-dried for two weeks. The dried plants' materials were further chopped into smaller pieces and grounded to powder. The powdered material was soaked in 70% ethanol for 72 hr. The liquid filtrates were concentrated and evaporated to dryness in vacuo at 40˚C using rotary evaporator (Okokon et al., 2006).

Animals
Swiss albino rats of either sex (190 -220 g) used for these experiments were obtained from University of Uyo animal house. The animals were housed in standard cages and maintained on a standard pelleted feed (Guinea feed) and water was ad libitum. Permission and approval for animal studies were obtained from College of Health Sciences Animal Ethics committee, University of Uyo (UU/CHSAE/14/012).

Animal treatment
A total of 36 rats of both sexes were weighed and divided into six groups of 6 animals each and treated as follows: Group A consisted of normal animals that were administered with distilled water (0.2 ml/kg), Group B was administered with distilled water 0.2 ml/kg, while groups C, D and E were administered with p.o 30, 60 and 90 mg/kg/day of M. africana stembark extract (MASBEX), for 8 days, respectively. Group F was treated with silymarin (100 mg/kg) (standard/reference drug representing positive control) for the same period of time. Paracetamol, 2 g/kg, was administered to groups B -F on the eighth day. Twenty-four hours after paracetamol administration, the animals were sacrificed under light diethyl ether vapor. Blood were collected by cardiac puncture and used immediately.

Hematological study
Animals were sacrificed under diethyl ether anaesthesia, blood samples were collected from each rat by cardiac puncture using 21 gauge (21 G) needles mounted on a 5 ml syringe into ethylene diamine tetraacetic acid (EDTA) -coated sample bottles for analysis. Hematological parameters such as full blood count (FBC), hemoglobin, (Hb), packed cell volume (PCV), platelet concentration (PLC) and total and differential white blood cell count (WBC) were recorded. These parameters were analyzed using automatic hematological system (Sysmex Hematology -Coagulation system, Model MO-1000 I, Trans Asia, Japan).

Evaluation of the protective effect of the extract against paracetamol-induced liver injury regarding biochemical parameters and histology of liver of rats
Serum was separated from the blood samples and the sera were stored at -20 o C until used for biochemical determinations such as total protein, albumin, aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase(ALP), total cholesterol, total and direct bilirubin. The determinations were done spectrophotometrically using Randox analytical kits according to standard procedures of manufacturer's protocols (Reitman and Frankel, 1957). The livers of the animals were surgically removed, weighed and a part of each was fixed in 10% formaldehyde for histological processes, and the rest was washed with ice cold 0.9% NaCl and homogenates were made at a ratio of 1 g of wet tissue to 9 ml of 1.25% KCl using motor driven Teflonpestle. The homogenates were centrifuged at 7000 rpm for 10 min at 4˚C and the supernatants were used for the assays of superoxide dismutase (SOD) (Marklund et al., 1974), catalase (CAT) (Sinha, 1972), glutathione peroxidase (GPX) (Lawrence and Burk, 1976), and reduced glutathione (GSH) (Ellman, 1959).

Statistical analysis
Data obtained from this work were analyzed statistically using Student's t-test and ANOVA (One -way) followed by a post test (Tukey-Kramer multiple comparison test). Differences between means were considered significant at p < 0.05.

Results
Effect of ethanolic extract of M. africana stembark on the hematological parameters of rats with paracetamolinduced hepatotoxicity Administration of paracetamol (2 g/kg) to rats did not affect (p<0.05) RBC and WBC counts as well as PCV percentage and hemoglobin concentration significantly (Table 2). However, there were significant (p<0.001) increases in the percentages of neutrophils, while pretreatment with M. africana stembark extract did not affect the increases induced by paracetamol. There were significant (p<0.05-0.001) reductions in the percentages of lymphocytes, monocytes, eosinophils and basophils following paracetamol administration. Further significant (p<0.01) reductions in the number of monocytes and eosinophils were observed in the extract/silymarintreated rats ( Table 1).

Evaluation of the effect of M. africana stembark on liver function test of paracetamol-intoxicated rats
Administration of paracetamol (2 g/kg) to rats caused a significant (p<0.001) elevation of the levels of AST, ALT, ALP, total cholesterol, total and direct bilirubin and decrease in total protein and albumin levels when compared to control.  There were significant (p<0.01 -0.001) decreases in these enzymes levels and the level of total cholesterol, total and direct bilirubin in the groups pre-administered with the stembark extract of M. africana (30-90 mg/kg, groups C to E) when compared with the paracetamol group, which were dose-dependent. Total protein and albumin levels were significantly (p<0.05-0.001) elevated in a dosedependent manner in the groups pretreated with the stembark extract when compared to the paracetamol group. The effects of the highest dose of the extract on all of the parameters evaluated were comparable to those of silymarin ( Table  2).

Effect of stembark extract on liver weight
Liver weights of rats treated with paracetamol were significantly (p<0.001) increased when compared to those of the control group. However, animals in groups pre-treated with the stembark extract and silymarin showed a significant (p<0.01-0.001) decrease in weight when compared to control (Table 3).

Effect of stembark extract on the levels of liver antioxidant enzymes
Paracetamol treatment caused significant (p<0.001) decreases in the activities of SOD, CAT, GPx and GSH level in liver tissue when compared with control group (Table 4). Pre-treatment with stembark extract of M. africana (30 -90 mg/kg) resulted in a significant (p< 0.05-0.001) increase in the activities of SOD, CAT, GPx and GSH level. Silymarintreated animals also showed a significant (p< 0.001) increase in antioxidant enzymes (SOD, CAT, and GPx) and GSH levels compared to paracetamol-treated rats (Table 4).

Histopathological studies of rats liver with paracetamol -induced hepatotoxicity
Histopathological examination of liver sections of normal control group showed normal cellular architecture with distinct hepatic cells, sinusoidal spaces and central vein (Figures 1 A and B).
Disarrangement of normal hepatic cells with centrilobular necrosis, hyperplasia, vascular and cellular degeneration, polymorphonuclear aggregation, inflammation and fatty degeneration were observed in paracetamol-treated rats of group B (Figures 1 C and D).
The liver sections of the rats treated with stembark extract of M. africana (30 -90 mg/kg) of groups C, D and E showed signs of protection evidenced by the reduction/ absence of inflammatory cells, vascular congestion and degeneration, cellular degeneration, necrosis and vacuoles (Figure 1 C -J), while the liver sections of the rats treated with silymarin (100 mg/kg) in group F showed significant reduction in fatty degeneration and absence of necrosis and inflammation (Figures 1 K and L).

Discussion
In this study, paracetamol administration was found to cause elevation of enzymes levels such as AST, ALT, ALP, total cholesterol, total and direct bilirubin and decrease total protein and albumin levels. These conditions were however reversed by pre-treatment with the extract and standard drug, silymarin. Paracetamol is reported to produce acute toxic effect at high doses which leads to liver damage as a result of its bioactivation by the cytochrome P450 system to a toxic electrophile, N-acetyl p-benzoquinone imine (NAPQI), which covalently binds to tissue macromolecules, probably oxidizes lipids or the critical sulfhydryl groups (protein thiols) and alters the homeostasis of calcium (Davern et al., 2006;Das and Sarma, 2009;Marotta et al., 2009;Jaeschkea and Bajt, 2010). This could have been the case in this study.
The massive production of reactive species may lead to depletion of physiological protective moieties (glutathione, α-tocopherol, etc.), ensuing wide-spread propagation of the alkylation and peroxidation, and damaging the macromolecules in vital biomembranes (Gilani et al., 2005). Induction of cytochrome and depletion of glutathione are the major predisposing factors to liver injury (Gupta et al., 2006).
Damage to the structural integrity of liver is reflected by an increase in the levels of serum transaminases AST, ALT and ALP, because they are cytoplasmic in location and are released into the circulation after cellular damage. Assessment of liver function can be undertaken by estimating the activities of serum ALT, AST, ALP, bilirubin (total and direct), total cholesterol, total protein and albumin which are originally present in the cytoplasm (Manokaran et al., 2008). When there is hepatopathy, these enzymes and molecules leak into the blood stream serving as an indicator of the liver damage (Nkosi et al., 2005). The aminotransferases (ALT and AST) are the most commonly used specific indicators of hepatic necrosis (Dama et al., 2011). ALT is a sensitive indicator of acute liver damage and elevation of this enzyme in nonhepatic diseases is unusual. ALT is more selectively a liver paranchymal enzyme than AST (Nkosi et al., 2005).
Abnormally high levels of serum ALT, AST, ALP, total bilirubin (total and direct), and total cholesterol and decreases in total protein and albumin levels observed in paracetamol group in our study are the consequence of paracetamolinduced liver dysfunction and denote the damage to the hepatic cells due to paracetamol intoxication (Parmar et al., 2010;Mandade, 2011).
Serum ALP, bilirubin and total protein levels are related to the function of hepatic cell. Elevated level of ALP suggests biliary damage or an obstruction of the biliary tree, which disrupts the flow of blood to the liver (Kouitchev et al., 2007;Farida et al., 2012). The reversal of increased serum enzymes in paracetamolinduced liver damage by the extract may be due to the prevention of the leakage of intracellular enzymes by its membrane stabilizing activity.
In the present study, reduction in serum total protein and albumin levels were observed in paracetamol-treated rats which may be associated with the decrease in the number of hepatocytes which in turn may result in a decrease in hepatic capacity to synthesize protein and albumin. Hypoalbuminemia portrays an advanced chronic liver diseases and a reduction in serum total protein level is a useful index of the severity of cellular dysfunction in chronic liver diseases. Decreased levels of total protein and albumin as recorded in paracetamol-treated rats revealed the severity of hepatopathy and may be attributed to the damage produced and localized in the endoplasmic reticulum leading to decrease in protein synthesis (Kanchana and Sadiq, 2010). This negative effect on total protein and albumin was reversed in extract-pretreated groups and is clear indication of the improvement of the functional status of the liver cells by the extract.
Bilirubin, a metabolic product of hemoglobin, undergoes conjugation with glucuronic acid in hepatocytes to increase its water solubility. Elevated levels of bilirubin due to paracetamol intoxication in this study may be attributed to excessive heme destruction and blockage of biliary tract resulting in inhibition of the conjugation reaction and release of unconjugated bilirubin from damaged hepatocytes (Ali et al., 2010). Decrease in serum bilirubin after treatment with the extract in liver with paracetamol-induced damage, indicated the effectiveness of the extract to restore normal functional status of the liver.
Hepatotoxic substances are known to cause impairment of cholesterol metabolism leading to an increase in serum levels of cholesterol causing fatty liver (Farida et al., 2012). Paracetamol-induced toxicity in rats may have altered membrane structure and function as well as lipids metabolism in the liver as suggested by increased cholesterol levels in rats. This result is an indication of membrane rigidity caused by paracetamol. This effect was reduced by the protective activity of the stembark extract which restored the level of total cholesterol to near normal.
Superoxide dismutase, one of the most important intracellular antioxidant enzymes present in all aerobic cells, has an anti-toxic effect against superoxide H anion (Loki and Rajamohan, 2003). It scavenges the superoxide anion to form hydrogen peroxide, hence reducing the toxic effects caused by these radicals. In this study, it was observed that M. africana stembark extract significantly increased hepatic SOD activity in paracetamolinduced liver damage in rats. This showed that M. africana can reduce reactive free radicals, thereby reducing oxidative damage to the tissues besides improving activity of hepatic antioxidant enzymes.
Catalase is a haemoprotein that protects cells from the accumulation of H 2 O 2 by reducing it to form H 2 O and O 2 or by using it as an oxidant in which it works as peroxidase.
Therefore, decreased CAT activity may result in a number of deleterious effects due to the inability to scavenge superoxide radical and hydrogen peroxide. However, the activities of SOD and CAT in the liver were significantly reduced in paracetamol-treated rats than the normal rats. This effect suggests excessive formation of free radicals and activation of lipid peroxidation system resulting in tissue damages (Kuriakose and Kurup, 2008). Doses of the stembark extract (30 -90 mg/kg) increases the level of CAT in a similar manner as silymarin, the standard hepatoprotective drug, revealing its potentials to prevent the accumulation of excessive free radicals, thus protecting the liver from paracetamol intoxication.
Glutathione (GSH) is one of the tripeptide and nonenzymatic biological antioxidants that is present in high quantity in the liver. It helps to remove free radical species such as hydrogen peroxide, superoxide radicals, alkoxy radicals and maintain membrane protein thiols, through GPx and GST activities and maintenance of membrane protein thiols (Prakash et al., 2001;Mandade, 2011). Determination of total GSH is a key factor to show the antioxidant reserve in the organism (Odukoya et al, 2007;Balouchxadehet al 2011). Excessive peroxidation causes increased GSH consumption. GSH is a scavenger of toxic metabolites, including NAPQI, which is a metabolite of APAP (Hwang et al., 2008;Yuan et al., 2010). Reduced level of GSH is implicated in the enhancement of lipid peroxidation in paracetamol-treated rats (Parmer et al., 2010). Administration of M. africana and silymarin significantly increased the level of GPx and GSH in a dose-dependent manner portraying its ability to scavenge these free radicals. The activities of the extract and silymarin is in agreement with a number of previous studies on hepatoprotective effect of plant extract and silymarin (Oyegbami and Odetola, 2010;Zamani-Moghaddani et al., 2012).
Silymarin, a mixture of flavonolignans consisting of silybin, silychristin, silydianin and isosilybin, is a standardized seed extract of Silybum marianum (Madani et al, 2008) .Studies in cell culture and animal models clearly show its hepatoprotective property against carbon tetrachloride, paracetamol, Amanita phalloide toxin and thioacetamide (Muriel and Mourelle, 1990;Muriel et al., 1992;Das, 2012). Silymarin at doses up to 100 mg/kg has been used as a standard hepatoprotective agent by numerous investigators and it exerts hepatoprotective effect due to its antioxidant and scavenging properties (Kosina et al., 2002).
Certain drugs including alkylating cytotoxic agents could also affect blood formation rate and hematological parameters (Adeneye et al., 2008). Administration of paracetamol to rats increases erythrocyte membrane peroxidation, which may also lead to haemolytic changes. Treatment of rats with paracetamol in this study, did not significantly affect the haematological parameters such as RBC, Hb, PCV, and WBC levels except increases in neutrophils percentage and reductions in the percentages of monocytes, eosinophils and basophils. Pre-administration of M. africana extract to these animals also did not affect the above states though further reductions were observed in the percentages of monocytes and eosinophils as compared to the paracetamol-treated group.
The histological findings corroborate that of the biochemical results as the degree of injury induced by paracetamol was reduced significantly by pretreatment with the stembark extract.
The stembark extract and fractions have been reported to exhibit strong cellular antioxidant activity in whole blood, neutrophils (extracellular and intracellular) and macrophages (Okokon et al., 2013). Similarly, the cytotoxic coumarins isolated from the stembark have also been reported to exert strong antioxidant activity (Nguelefack-Mbuyo et al., 2010). This activity demonstrates the potential of the extract to inhibit reactive oxygen species (ROS) and scavenge free radicals like superoxide, hydrogen peroxide, etc. which can be attributed to the presence of coumarins and other phenolic compounds in the stembark what were reported earlier (Carpenter et al 1971;Games, 1972;Crichton and Waterman, 1978;Ouahouo et al., 2004;Nguelefack-Mbuyo et al., 2010;Okokon et al., 2013). The strong antioxidant activity of this extract explains the significant hepatoprotective activity of the stembark extract. Free radical mediated process has been associated with pathogenesis of most of the diseases. The activities of antioxidant counteract the redox state precipitated intracellularly and hence ensure hepatoprotection against paracetamol-induced liver injury. The antioxidant activity of this extract may as well explain the mechanism of action of the observed hepatoprotective activity of M. africana which may be related to inhibition of lipid peroxidation and enhancement of antioxidant enzyme levels in addition to free radicals scavenging action.
The study shows that Mammea africana possesses strong hepatoprotective activity which is due to its antioxidant activity precipitated by its chemical constituents. This confirms the use of M. africana stembark as an antidote in traditional medicine.